Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in Western Kenya.

OBJECTIVE: The antigen, falciparum malaria protein 1 (FMP1), represents the 42-kDa C-terminal fragment of merozoite surface protein-1 (MSP-1) of the 3D7 clone of P. falciparum. Formulated with AS02 (a proprietary Adjuvant System), it constitutes the FMP1/AS02 candidate malaria vaccine. We evaluated this vaccine's safety, immunogenicity, and efficacy in African children. METHODS: A randomised, double-blind, Phase IIb, comparator-controlled trial.The trial was conducted in 13 field stations of one mile radii within Kombewa Division, Nyanza Province, Western Kenya, an area of holoendemic transmission of P. falciparum. We enrolled 400 children aged 12-47 months in general good health.Children were randomised in a 1ratio1 fashion to receive either FMP1/AS02 (50 microg) or Rabipur(R) rabies vaccine. Vaccinations were administered on a 0, 1, and 2 month schedule. The primary study endpoint was time to first clinical episode of P. falciparum malaria (temperature >/=37.5 degrees C with asexual parasitaemia of >/=50,000 parasites/microL of blood) occurring between 14 days and six months after a third dose. Case detection was both active and passive. Safety and immunogenicity were evaluated for eight months after first immunisations; vaccine efficacy (VE) was measured over a six-month period following third vaccinations. RESULTS: 374 of 400 children received all three doses and completed six months of follow-up. FMP1/AS02 had a good safety profile and was well-tolerated but more reactogenic than the comparator. Geometric mean anti-MSP-1(42) antibody concentrations increased from1.3 microg/mL to 27.3 microg/mL in the FMP1/AS02 recipients, but were unchanged in controls. 97 children in the FMP1/AS02 group and 98 controls had a primary endpoint episode. Overall VE was 5.1% (95% CI: -26% to +28%; p-value = 0.7). CONCLUSIONS: FMP1/AS02 is not a promising candidate for further development as a monovalent malaria vaccine. Future MSP-1(42) vaccine development should focus on other formulations and antigen constructs. TRIAL REGISTRATION: Clinicaltrials.gov NCT00223990.

Protection induced by Plasmodium falciparum MSP1(42) is strain-specific, antigen and adjuvant dependent, and correlates with antibody responses.

Vaccination with Plasmodium falciparum MSP1(42)/complete Freund's adjuvant (FA) followed by MSP1(42)/incomplete FA is the only known regimen that protects Aotus nancymaae monkeys against infection by erythrocytic stage malaria parasites. The role of adjuvant is not defined; however complete FA cannot be used in humans. In rodent models, immunity is strain-specific. We vaccinated Aotus monkeys with the FVO or 3D7 alleles of MSP1(42) expressed in Escherichia coli or with the FVO allele expressed in baculovirus (bv) combined with complete and incomplete FA, Montanide ISA-720 (ISA-720) or AS02A. Challenge with FVO strain P. falciparum showed that suppression of cumulative day 11 parasitemia was strain-specific and could be induced by E. coli expressed MSP1(42) in combination with FA or ISA-720 but not with AS02A. The coli42-FVO antigen induced a stronger protective effect than the bv42-FVO antigen, and FA induced a stronger protective effect than ISA-720. ELISA antibody (Ab) responses at day of challenge (DOC) were strain-specific and correlated inversely with c-day 11 parasitemia (r = -0.843). ELISA Ab levels at DOC meeting a titer of at least 115,000 ELISA Ab units identified the vaccinees not requiring treatment (noTx) with a true positive rate of 83.3% and false positive rate of 14.3 %. Correlation between functional growth inhibitory Ab levels (GIA) and cumulative day 11 parasitemia was weaker (r = -0.511), and was not as predictive for a response of noTx. The lowest false positive rate for GIA was 30% when requiring a true positive rate of 83.3%. These inhibition results along with those showing that antigen/FA combinations induced a stronger protective immunity than antigen/ISA-720 or antigen/AS02 combinations are consistent with protection as ascribed to MSP1-specific cytophilic antibodies. Development of an effective MSP1(42) vaccine against erythrocytic stage P. falciparum infection will depend not only on antigen quality, but also upon the selection of an optimal adjuvant component.

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host.

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.

Phase 1 randomized double-blind safety and immunogenicity trial of Plasmodium falciparum malaria merozoite surface protein FMP1 vaccine, adjuvanted with AS02A, in adults in western Kenya.

We report the first trial of candidate malaria vaccine antigen FMP1, a 42kDa fragment from the C-terminus of merozoite surface protein-1 (MSP-1) from the 3D7 strain of Plasmodium falciparum, in an endemic area. Forty adult male and female residents of western Kenya were enrolled to receive 3 doses of either FMP1/AS02A or Imovax rabies vaccine by intra-deltoid injection on a 0, 1, 2 month schedule. Thirty-seven volunteers received all three immunizations and 38 completed the 12-month evaluation period. Slightly more recipients of the FMP1/AS02A vaccine experienced any instance of pain at 24 h post-immunization than in the Imovax group (95% versus 65%), but otherwise the two vaccines were equally safe and well-tolerated. Baseline antibody levels were high in both groups and were boosted in the FMP1/AS02A group. Longitudinal models revealed a highly significant difference between groups for both the average post-baseline antibody responses to MSP-1(42) (F1,335=13.16; P<0.001) and the Day 90 responses to MSP-1(42) (F1,335=16.69; P<0.001). The FMP1/AS02A vaccine is safe and immunogenic in adults and should progress to safety testing in children at greatest risk of malaria.

Safety and reactogenicity of an MSP-1 malaria vaccine candidate: a randomized phase Ib dose-escalation trial in Kenyan children.

OBJECTIVE: Our aim was to evaluate the safety, reactogenicity, and immunogenicity of an investigational malaria vaccine. DESIGN: This was an age-stratified phase Ib, double-blind, randomized, controlled, dose-escalation trial. Children were recruited into one of three cohorts (dosage groups) and randomized in 2:1 fashion to receive either the test product or a comparator. SETTING: The study was conducted in a rural population in Kombewa Division, western Kenya. PARTICIPANTS: Subjects were 135 children, aged 12-47 mo. INTERVENTIONS: Subjects received 10, 25, or 50 microg of falciparum malaria protein 1 (FMP1) formulated in 100, 250, and 500 microL, respectively, of AS02A, or they received a comparator (Imovax (rabies vaccine). OUTCOME MEASURES: We performed safety and reactogenicity parameters and assessment of adverse events during solicited (7 d) and unsolicited (30 d) periods after each vaccination. Serious adverse events were monitored for 6 mo after the last vaccination. RESULTS: Both vaccines were safe and well tolerated. FMP1/AS02A recipients experienced significantly more pain and injection-site swelling with a dose-effect relationship. Systemic reactogenicity was low at all dose levels. Hemoglobin levels remained stable and similar across arms. Baseline geometric mean titers were comparable in all groups. Anti-FMP1 antibody titers increased in a dose-dependent manner in subjects receiving FMP1/AS02A; no increase in anti-FMP1 titers occurred in subjects who received the comparator. By study end, subjects who received either 25 or 50 microg of FMP1 had similar antibody levels, which remained significantly higher than that of those who received the comparator or 10 microg of FMP1. A longitudinal mixed effects model showed a statistically significant effect of dosage level on immune response (F(3,1047) = 10.78, or F(3, 995) = 11.22, p < 0.001); however, the comparison of 25 microg and 50 microg recipients indicated no significant difference (F(1,1047) = 0.05; p = 0.82). CONCLUSIONS: The FMP1/AS02A vaccine was safe and immunogenic in malaria-exposed 12- to 47-mo-old children and the magnitude of immune response of the 25 and 50 microg doses was superior to that of the 10 microg dose.

Critical evaluation of different methods for measuring the functional activity of antibodies against malaria blood stage antigens.

Antibodies are thought to be the primary immune effectors in the defense against erythrocytic stage Plasmodium falciparum. Thus, malaria vaccines directed to blood stages of infection are evaluated based on their ability to induce antibodies with anti-parasite activity. Such antibodies may have different effector functions (e.g., inhibition of invasion or inhibition of parasite growth/development) depending on the target antigen. We evaluated four methods with regards to their ability to differentiate between invasion and/or growth inhibitory activities of antibodies specific for two distinct blood stage antigens: AMA1 and MSP1(42). We conclude that antibodies induced by these vaccine candidates have different modes of action that vary not only by the antigen, but also by the strain of parasite being tested. Analysis based on parasitemia and viability was essential for defining the full range of anti-parasite activities in immune sera.

Heterologous prime-boost immunization in rhesus macaques by two, optimally spaced particle-mediated epidermal deliveries of Plasmodium falciparum circumsporozoite protein-encoding DNA, followed by intramuscular RTS,S/AS02A.

BACKGROUND: RTS,S/AS02A, a recombinant Plasmodium falciparum vaccine based on the circumsporozoite protein (CSP) repeat and C-terminus regions, elicits strong humoral and Th1 cell-mediated immunity. In field studies, RTS,S/AS02A reduced malaria infection, clinical episodes, and disease severity. Heterologous prime-boost immunization regimens, optimally spaced, might improve the protective immunity of RTS,S/AS02A. METHODS: DNA plasmid encoding P. falciparum CSP (3D7) was administered to six experimental groups of rhesus monkeys (N = 5) by gene gun (coded as D), followed by a 1/5th human dose of RTS,S/AS02A (coded as R). Immunization regimens, including a numeral to denote weeks between immunizations, were D-4-R, D-16-R, D-4-D-4-R, D-4-D-16-R, D-16-D-4-R and D-16-D-16-R. A control group (N = 5) received a single 1/5th dose of RTS,S/AS02A. Endpoints were antibody (Ab) to homologous CSP repeat and C-terminus regions and delayed-type hypersensitivity (DTH) to CSP peptides. FINDINGS: Monkeys immunized twice with DNA, 16 weeks apart (D-16-D-4-R and D-16-D-16-R), developed higher levels of anti-C-terminus Abs than control monkeys (p<0.02). No CSP DNA priming regimen increased RTS,S/AS02A-induced Ab to CSP repeats. At 16 months after first immunization, D-R and D-D-R, but not control, monkeys had histologically confirmed DTH reactions against CSP C-terminus, which persisted at repeat testing 12 months later. INTERPRETATION: Two optimally spaced, particle-mediated epidermal deliveries of CSP DNA improved the humoral immunogenicity of a single dose of RTS,S/AS02A. Further, CSP DNA prime followed by one dose of RTS,S/AS02A gave biopsy proven DTH reactions against CSP C-terminus of up to 2 years duration, implying the induction of CD4+ memory T cells. Heterologous prime-boost strategies for malaria involving gene gun delivered DNA or more potent vectors, administered at optimal intervals, warrant further investigation.

Phase I safety and immunogenicity trial of FMP1/AS02A, a Plasmodium falciparum MSP-1 asexual blood stage vaccine.

We report the first safety and immunogenicity trial of the Plasmodium falciparum malaria blood stage vaccine candidate, FMP1/AS02A consisting of the FMP1 antigen, an Escherichia coli-expressed His-tagged fusion protein from the 42 kDa C-terminal fragment from the 3D7 clone of the merozoite surface protein 1 formulated in the AS02A adjuvant. An open label, prospective, single-center Phase I dose escalation trial of FMP1/AS02A was conducted in 15 adult malaria-naïve human volunteers to assess safety, reactogenicity, and immunogenicity. The vaccine was safe and well-tolerated and no serious adverse events were observed. The vaccine induced high-titer ELISA and IFA responses in all volunteers. Proliferative and ELISPOT responses were induced to vaccine antigen. Biologically active antibodies were induced as measured by GIA. This study establishes the foundation to further evaluate and measure the vaccine's ability to reduce morbidity and mortality in target populations directly affected by P. falciparum malaria.

Process development and analysis of liver-stage antigen 1, a preerythrocyte-stage protein-based vaccine for Plasmodium falciparum.

Plasmodium falciparum liver-stage antigen 1 (LSA-1) is expressed solely in infected hepatocytes and is thought to have a role in liver schizogony and merozoite release. Specific humoral, cellular, and cytokine immune responses to LSA-1 are well documented, with epitopes identified that correlate with antibody production, proliferative T-cell responses, or cytokine induction. With the goal of developing a vaccine against this preerythrocyte-stage protein, we undertook the good manufacturing practices (GMP) manufacture of a recombinant LSA-1 construct, LSA-NRC, incorporating the N- and C-terminal regions of the protein and two of the centrally placed 17-amino-acid repeats. To improve the protein yield, a method of codon harmonization was employed to reengineer the gene sequence for expression in Escherichia coli. A 300-liter GMP fermentation produced 8 kg of bacterial cell paste, and a three-step column chromatographic method yielded 8 mg of purified antigen per g of paste. The final bulk protein was >98% pure, demonstrated long-term stability, and contained <0.005 endotoxin units per 50 microg of protein. To accomplish the initial stages of evaluation of this protein as a human-use vaccine against malaria, we immunized rabbits and mice with LSA-NRC in Montanide ISA 720. New Zealand White rabbits and A/J (H-2K) mice produced high-titer antibodies that recognized liver-stage parasites in infected cultured human hepatocytes. Gamma interferon-producing cells, which have been associated with LSA-1-mediated protection, were detected in splenocytes harvested from immunized mice. Finally, sera taken from people living in a region where malaria is holoendemic recognized LSA-NRC by Western blotting.

Towards an RTS,S-based, multi-stage, multi-antigen vaccine against falciparum malaria: progress at the Walter Reed Army Institute of Research.

The goal of the Malaria Vaccine Program at the Walter Reed Army Institute of Research (WRAIR) is to develop a licensed multi-antigen, multi-stage vaccine against Plasmodium falciparum able to prevent all symptomatic manifestations of malaria by preventing parasitemia. A secondary goal is to limit disease in vaccinees that do develop malaria. Malaria prevention will be achieved by inducing humoral and cellular immunity against the pre-erythrocytic circumsporozoite protein (CSP) and the liver stage antigen-1 (LSA-1). The strategy to limit disease will target immune responses against one or more blood stage antigens, merozoite surface protein-1 (MSP-1) and apical merozoite antigen-1 (AMA-1). The induction of T- and B-cell memory to achieve a sustained vaccine response may additionally require immunization with an adenovirus vector such as adenovirus serotype 35. RTS,S, a CSP-derived antigen developed by GlaxoSmithKline Biologicals in collaboration with the Walter Reed Army Institute of Research over the past 17 years, is the cornerstone of our program. RTS,S formulated in AS02A (a GSK proprietary formulation) is the only vaccine candidate shown in field trials to prevent malaria and, in one instance, to limit disease severity. Our vaccine development plan requires proof of an individual antigen's efficacy in a Phase 2 laboratory challenge or field trial prior to its integration into an RTS,S-based, multi-antigen vaccine. Progress has been accelerated through extensive partnerships with industrial, academic, governmental, and non-governmental organizations. Recent safety, immunogenicity, and efficacy trials in the US and Africa are presented, as well as plans for the development of a multi-antigen vaccine.

C3d binding to the circumsporozoite protein carboxy-terminus deviates immunity against malaria.

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity.

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites.

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.

Pre-clinical evaluation of the malaria vaccine candidate P. falciparum MSP1(42) formulated with novel adjuvants or with alum.

We compared the safety and immunogenicity of the recombinant Plasmodium falciparum MSP1(42) antigen formulated with four novel adjuvant systems (AS01B, AS02A, AS05 and AS08) to alum in rhesus monkeys. All five formulations of MSP1(42) were safe and immunogenic. Whereas, all MSP1(42) formulations tested generated high stimulation indices for lymphocyte proliferation (ranging from 27 to 50), the AS02A and AS01B formulations induced the highest levels of specific anti-MSP1(42) antibody. ELISPOT assays showed that the AS02A and AS01B vaccine formulations-induced different cytokine response profiles. Using the ratio of IFN-gamma/IL-5 secreting cells as the metric, the AS01B formulation induced a strong Th1 response, whereas the AS02A formulation induced a balanced Th1/Th2 response. The IFN-gamma response generated by AS02A and AS01B formulations persisted at least 24 weeks after final vaccination. The notable difference in Th1/Th2 polarization induced by the AS02A and AS01B formulations warrants comparative clinical testing.

Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine.

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.